The proton-translocating NADH-quinone oxidoreductase is one of the four enzyme complexes in the respiratory chain of most aerobic species from bacteria to human. The enzyme is collectively called complex I and its bacterial counterpart is specifically referred to as NDH-1. Complex I/NDH-1 is recognized to have the most intricate structure of the membrane-associated enzyme complexes. Low resolution EM analyses indicate that the enzyme has an L-shaped structure consisting of a membrane domain and a peripheral domain. Recently, high resolution 3D structure of the peripheral domain of NDH-1 became available. Complex I/NDH-1 contains one FMN and eight or nine FeS clusters as cofactors. These cofactors are located in the peripheral domain. This enzyme pumps protons and builds an electrochemical gradient across the membrane. Mammalian complex I is located in the innermitochondrial membrane and is composed of at least 45 different subunits with a total molecular mass of approximately 1 MDa. It is known that structural and functional defects of complex I are involved in many human mitochondrial diseases. Furthermore, sporadic Parkinson's disease is also related to complex I deficiencies. In contrast to the mitochondrial enzyme, NDH-1 has a simpler structure and is composed of 14 subunits. Because of its structural simplicity and availability of the 3D structure of the peripheral segment, NDH-1 serves as a useful model system for studying the structure and function of the mammalian enzyme. There is another type of NADH-quinone oxidoreductase named NDH-2 that is not found in mammals. NDH-2 is composed of a single polypeptide and does not pump protons. We have demonstrated that the yeast Ndi1, a member of NDH-2, can protect animals against complex I deficiencies. The overall goal of this grant application is to elucidate the structure and the mechanism of action of complex I/NDH-1 and Ndi1. The studies planned for this grant period are as follows: (1) Identification and characterization of the essential amino acid residues in the membrane domain subunits of NDH-1;(2) Clarification of functional roles of FeS clusters of NDH-1;(3) Identification of the Q-binding site and the inhibitor-binding site of NDH-1;(4) Clarification of the structure and the mechanism of action of the yeast Ndi1. Defects of mitochondrial NADH dehydrogenase cause many human diseases including sporadic Parkinson's disease. In order to develop therapies for diseases caused by deficiencies of this enzyme, it is a prerequisite to investigate why and how defects occur.